Human cytomegalovirus, the cause of serious infections in immunocompromised individuals, encodes several functions that block programed cell death. These functions are likely to play important roles in viral replication and pathogenesis by modulating the intrinsic impact of the virus on the host cell as well as by counteracting extrinsic signals of apoptosis that arise as a result of innate and adaptive clearance mechanisms mounted by the host. This continuation of research on CMV replication will focus on three genes encoding anti-apoptotic CMV functions: UL37, a viral mitochondrial-localized inhibitor (vMIA), UL36, a viral caspase 8 inhibitor (vlCA), and UL144, a TNF receptor homolog that may disrupt signalling. This project will investigate roles for these gene products preventing (1) intrinsic, stress-related induction of apoptosis that results from expression of viral functions in the course of productive replication, and (2) extrinsic signals such as those resulting from the immune response, including engagement of death domain-signalling receptors or introduction of pro-apoptotic granzymes. The two most potent viral inhibitors, pUL37x1/vMIA and gpUL36/vlCA, will be subjected to detailed mutagenesis to map specific domains that contact cellular targets. Minimal-size, fully active versions of each will be made and tested in the context of the virus for activity. The pro-apoptotic signals induced during infection will be evaluated to identify whether virion structural or immediate early gene products, such as the regulatory gene IE2-p86, trigger apoptosis. A role for UL37, UL36 or UL144 gene products in preventing apoptosis or other aspects of viral replication will be investigated by performing microarray analysis using viral mutants to assay changes in expression of 43,000 human cDNAs representing all human CMV genes. Evaluation of these functions will be carried out in cell culture with human CMV mutants and in infected mice using murine CMV, either by mutating the gene homolog or by replacing/adding the human CMV function to the murine CMV genome. The role of these functions in evading cytokine/death receptor or cell-mediated immune responses will be determined in strains of mice lacking relevant effector mechanisms. Through these efforts we expect to gain a complete understanding of the role of CMV genes that promote efficient replication or deflect the host immune response by preventing host cell apoptosis.